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Recombinant DNA

Hi,

I was hoping someone could help me.

If you know anything at all about recombinant DNA, how it is formed and how it works could you please explain it to me (simply lol :smile:

). I have been on a few sites and am majorly confused :confused:

. I am trying to write about how it is used to produce antibodies.

Thanks in advance :smile:

.

luv from me. XXX

Reply 1

acvaos

hi there, recombinatant dna is simply dna which has been genetically engineered to contain DNA from another species. For what you want to write about you need to talk prinicipally about genetic engineering one would presume.

Reply 2

Tom H

Naturally occuring antibodies or engineered antibodies?

If you're talking about naturally occuring antibodies, then that's done via a process called somatic recombination. Basically enzymes, mainly the enzymes RAG1 and RAG2 (the RAG recombinases) bend and chop out DNA to create unique DNA sequences that represent unique antibodies.

If you want to know more about that, have a google of sommatic recombination.

If you're talking about the generation of engineered antibodies, there are various different methods to do it. However most involve the basics of cloning, PCR, restriction digests and ligations.

Reply 3

Wangers

OP - when you say recombinant antibodies,

do you mean Monoclonal antibodies - these are a B lymphacyte fused with a immortal cancerous cell - look up Paraprotein, and the work of Milsein and Jerne.

Or do you mean Chimeric antibodies - ie using animals to produce antibodies ( I think its Chimeric if its 75% animal). Although animal antibodies can now be made to bear human protein recognition keys I believe.

Reply 4

Tom H

If you're talking about monoclonals then there are several different types as Wangers says.

1. Animal monoclonal antibodies

Usually of mouse origin. Mice are immunised with the antigen of choice and then high affinity antibodies are produced by isolating B-lymphocytes and immortalising them.

However using these antibodies in humans presents problems, as they are from mouse they illicit an immune response in humans. Which can be potentially fatal.

2. Chimeric Antibodies

As the fact the antibodies are of mouse origin is what's causing the problem, chimeric antibodies hope to reduce this by cutting and pasting the variable regions of the mouse antibody (which contain its binding activity) onto human constant regions. This is where some recombinant DNA tech is used, presumably fusion PCR.

3. Humanised Antibodies

Chimeric antibodies still have a large area that is of mouse origin. However, looking at the variable regions only certain residies within regions called complimentarity determining regions (CDRs) actually contain the binding activity. Therefore, using human antibodies as a frame work, these residues replace the ones in the human antibody.

I guess the DNA tech used here is more Site Directed Mutagenesis or casette mutagenesis.

4. Human antibodies

Despite the drastic reduction in mouse residues, humanised antibodies still have some. The solution is to make fully human antibodies, with no mouse residues involved at all. Now this, for ethical reasons, can't be done by immunising humans with the antigen. Instead huge DNA libraries of millions of potetinal antibodies have been created. These then need to be screened against the antigen to see which ones bind the target. This is done via display technologies such as Ribosome Display, Bacterial Display and Phage Display that couple phenotype (the antibody and its ability to bind/not bind) with its genotype (the DNA sequence encoding that antibody)

Reply 5

acvaos

wouldnt there have to be a reference to restriction endonuclease.

Reply 6

31Jen16

Hi guys, Thanks for all your help :smile:

The notes i have talk about monoclonal DNA and recombinant DNA producing antibodies by different processes.

I have written a paragraph about monoclonal and have started to do one about recombinant. As far as I understand it (recombinant) is just two peices of DNA joined together and placed in a host cell which under the right conditions will reproduce itself to give many antibodies. One thing I really don't understand is which two peices of DNA are used. Is one from a mouse that has been injected with antigens?? If so what is the other peice?

I am so sorry if I sound really thick :confused:

. Any help will be appreciated :smile:

Reply 7

Wangers

Yes recombinant is a forign piece of DNA introduced to the normal sequence. I think your syllabus only requires Restriction Endonucleases - which cut to "Sticky ends". and then Ligase - which forms phosphodiester bonds.

To test if it has combined - you introduce it across a second antibiotic resistance gene, then use the principle of replica plating. Or you can also use phospholuminesscence. The recombinant bit can be any DNA you want to introduce, as long as it contains a priming sequence and a replication point.

Then introduce the plasmid to the new host - uou could use tempreture shock methods, or you could use salt bath I think.

I think you're getting slightly confused in the second bit there -

What your thinking is Monoclonal Antibodies. Look up Milstein and Jerne. Basiclly a cancerous B cell can only produce one type of antibody - and all its future generations will only produce that antibody. It has lost the ability to adapt and specialise. You then combine that with a B cell that produces the best antibody for a certain antigen. And you have a cell that will endlesly produce the antibody you want. Because it retains some cancerous properties, it is said to be immortal in cell culture.

Look up HPGRT salvage pathway. This pathway, combined with clever use of an inhibitor allows selection of succssfully dused cells. Initially Hetra and even Polykaryotes form, and DNA is ejected until the correct number of Chromosomes are left. Note that this has to be a functional set. So therefore its actually quite a costly procedure to make Mabs.

Since the two are fused - its whole DNA that is input, also the effective portion is a tiny fraction of that.

MAbs are now leading the way - you may have heard of Tamiflu - the anti bird fly drug that the goverment is stockpiling. There is also Herceptin - the drug that caused furor in the "postcode lottary" accusations.

Reply 8

Tom H

I think there is a lot of confusion here. Are we talking about commercially engineered mAbs or naturally occuring antibodies?

Genetic recombination doesn't necessarily have to be two bits of DNA added together. It's just the term for DNA that has been recombined to give a sequence that is different from what you had originally.

In naturally occuring antibodies, the antibody genes naturally undergo a recombination (sommatic recombination), which generates millions of different DNA sequences that each produce a different antibody.

In engineered antibodys, recombination is used more in the artifical sense. In pure mouse antibodies there is very little recombination needed other than splicing the seqence into a mammalian expression vector.